Campylobacter Colitis: Histological Immuno- Histochemical and Ultrastructural Findings

Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from

Gut, 1985, 26, 945-951

Campylobacter colitis: histological immunohistochemical and ultrastructural findings

J P VAN SPREEUWEL, G C DUURSMA, C J L M MEIJER, R BAX, P C M ROSEKRANS, AND J LINDEMAN From the Department ofInternal Medicine, Division of Gastroenterology, St. Antonius Ziekenhuis, Nieuwegein, Department ofMicrobiology and Pathology, Stichting Samenwerking Delftse Ziekenhuizen, Delft, Department ofInternal Medicine, St. Elizabeth Ziekenhuis, Leiderdorp, and the Department of Pathology, Vrije Universiteit, Amsterdam, The Netherlands

SUMMARY The colonic biopsy specimens of 22 patients with colitis and positive stool cultures for Campylobacter jejuni were studied in order to obtain histological and immunohistochemical criteria to differentiate Campylobacter colitis from chronic inflammatory bowel disease. In addition we tried to identify Campylobacter inclusions by means of immunohistochemistry and electron microscopy as evidence for invasion of the colonic mucosa. The results show that the majority of patients with Campylobacter colitis have the histological picture of acute infectious colitis with increased numbers of IgA and IgM containing plasma cells in the colonic mucosa in contrast with patients with active chronic inflammatory bowel disease who show increases of IgA and IgG (ulcerative colitis) or IgA-, IgM and IgG containing plasma cells (M Crohn) in their colonic biopsies. The results of immunohistochemical stainings with Campylobacter antiserum show invasion of Campylobacter in the colonic mucosa. These findings were confirmed ultrastructurally. http://gut.bmj.com/

In the past decade Campylobacter has emerged be indistinguishable from ulcerative colitis. Histo- from obscurity to become an important human logical examination of rectal biopsies has ranged pathogen. Provided that proper techniques are from normal to inflammatory changes suggestive of applied Campylobacter fetus subspecies jejuni can acute infectious colitis or ulcerative colitis or

be isolated from the stools of patients with diarrhoea Crohn's disease.61' In the present study we have on September 30, 2021 by guest. Protected copyright. as often as Salmonella.1 2 In the laboratory for investigated the colonic biopsies of 22 patients with microbiology of the SSDZ, Delft, Campylobacter diarrhoea and stool cultures positive for Campylo- jejuni was isolated as the causative organism in 41% bacter jejuni. The aim of our study was to determine of 732 consecutive positive stool cultures of patients the histological features and the number of immuno- with diarrhoea. Campylobacter may cause a spec- globulin containing cells in colonic biopsies of trum of intestinal disease ranging from acute gas- patients with Campylobacter colitis and to identify troenteritis to toxic megacolon, lymphadenitis Campylobacter inclusions immunohistochemically. mesenterialis, and even appendicitis and cholecystitis.35 Usually infection with this organism results in Methods acute gastroenteritis with fever and frequent loss of often bloody stools. The disease resolves spon- PATIENTS taneously within one week but in 20% it runs a more One hundred and twelve colonic biopsies from 22 prolonged course or relapses resembling chronic patients with positive stool cultures for Campylobac- inflammatory bowel disease.1 Endoscopically it may ter jejuni were examined. One patient had been suffering from chronic ulcerative colitis for many years whereas one patient had previously suffered Address for correspondence: Dr J P van Spreeuwel, Department of Internal from non-specific proctitis. The others had no Medicine, Division of Gastroenterology, Ziekenhuis St. Annadal, St. Annadal 1, 6214 PA Maastricht, The Netherlands. coexisting gastrointestinal disease. Diarrhoea with Received for publication 1 November 1984 crampy abdominal pain was the presenting com- 945 Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from

946 van Spreeuwel, Duursma, Meijer, Bax, Rosekrans, and Lindeman plaint in 15 patients, 13 of them had noticed blood in 0-5 mm stretched muscularis mucosae. The latter is their stools, and fever was an accompanying symp- comparable with the 'mucosal tissue unit' described tom in seven of these 15 patients. Nausea was by Brandzaeg and Baklien.14 Moreover, immuno- recorded frequently. Six patients merely complained histochemistry was carried out with Campylobacter of loosing blood with their stools. One patient antiserum using both direct immunoperoxidase and presented with abdominal pain, nausea, and vomi- immunofluorescence techniques. The antiserum was tus. The numbers of immunoglobulin containing raised in a rabbit that was vaccinated intravenously cells in colonic biopsies of patients with Campylo- with a Campylobacter fetus subspecies jejuni bacter colitis were compared with those of 10 suspension containing microorganisms of serotype healthy controls, 10 patients with active Crohn's LAU1'5 that were killed in 1-5% formalin. Anti- disease of the colon and 10 patients with active serum titres were determined by agglutination and ulcerative colitis. All patients with chronic inflam- indirect immunofluorescence methods. After matory bowel disease had histologically abnormal several absorption steps with Salmonella g, Shigella colonic biopsies. boidii, Shigella sonnei, Proteus vulgaris and Escher- Stools were cultured for Salmonella, Shigella, ichia coli the antiserum showed no cross reactivity Yersinia and Campylobacter routinely. Campylo- with Salmonella, Shigella, or Escherichia coli. The bacter was cultured on a selective medium: blood antiserum gave a positive immunofluorescence with agar plates containing Campylobacter Growth Sup- 37 out of 38 different Campylobacter LAU sero- plement and Campylobacter Selective Supplement types; representing all known serotypes at that time (Oxoid LTD Basingstoke England nos. SR 84 and in the Netherlands. Immunofluorescence was SR 85). Inoculated plates were incubated during 48 achieved by removing paraffin with xylol and hours at 42°C in an atmosphere containing 5-15% alcohol, preincubation with normal swine serum 02 and 5-8% CO2 by using the GasPak of BBL 1:10, incubation with rabbit Campylobacter anti- without a catlyst in a jar. serum 1:40, once again incubation with normal Biopsies were fixed immediately either in a swine serum 1:10 and incubation with swine anti- formalin sublimate mixture for four to six hours for rabbit FITC. Sections stained with preimmune routine histology and immunohistochemical studies serum from the same rabbit served as controls. or in cacodylate buffered glutaraldehyde for ultra Colonic biopsies from eight patients with Crohn's structural studies. Tissue samples were embedded in disease of the colon and nine patients with ulcerative paraffin wax, cut in sections 4,um thick and mounted colitis stained with Campylobacter antiserum served http://gut.bmj.com/ on glass slides. Besides histological stains including as controls as well and were all negative. In addition haematoxylin and eosin and periodic acid Schiff, in three patients in whom immunohistochemistry biopsies were stained specifically for IgA, IgM, and with Campylobacter antiserum had given positive IgG heavy chains using an indirect immunoperox- results ultrastructural examination of the biopsies idase technique.12 Appropriate controls were done was done using a Zeiss EM 109 electron microscope. according to Sternberger.13 Rabbit antisera against Statistical analysis was carried out according to IgA, IgM, and IgG heavy chains were purchased the Student's t test, p<005 was considered signifi- from Dakopatts (Denmark). Their specificity was cant. on September 30, 2021 by guest. Protected copyright. confirmed by immunoelectrophoresis, immunofluorescence and immunoperoxidase stainings on Results bone marrow preparations monoclonal for IgA, IgM, or IgG. Horseradish peroxidase labelled goat The results are summarised in the Table. antirabbit Ig was obtained from Miles (Yedah, Israel). The IgA, IgM, and IgG stained sections HISTOLOGICAL FINDINGS were used for morphometrical analysis. They were In 19 patients the colonic biopsies showed a histolo- photographed with a standard magnification (100, 8 gical picture consistent with acute infectious colitis. x) and projected on a graphic tablet interfaced to a In the patient with longstanding ulcerative colitis computer (Ibas I Kontron Munchen). Every photo- with superimposed Campylobacter enterocolitis the graph contained approximately 1 mm mucosa at full histological features of ulcerative colitis dominated thickness. The lamina propria area was limited by with marked distortion of crypt architecture. In two two lines perpendicular to the muscularis mucosae patients histological examination revealed no abnor- and this area was measured per mm muscularis malities. Based on our findings in patients in whom mucosae length. The number of immunoglobulin follow up biopsies were obtained, and the time lapse containing cells was measured in two consecutive between the onset of bloody diarrhoea and the sections in approximately the same area and ex- biopsy we believe the histological changes of Cam- pressed both per 0 1 mm2 lamina propria and per pylobacter colitis can be divided in three stages. Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from

Campylobacter colitis: histological immunohistochemical and ultrastructural findings 947

Table Histological and immunohistochemical findings in 22 patients with positive stool culturesfor Campylobacterjejuni Immunohistochemistry Camp. antiserum Crypt surf Days after IgA IgM lgG epithelium onset of Histologicalfindings containing cells Glycocalyx lamina prop Histiocytes symptoms 1 Act. chronicinflam. T T T + + + ? Act. chronic inflam. T T T + + + 2 9 Acute colitis II T - - + 30 Normal + - 3 ? Acute colitis II T + - 4 ? Acute colitis II T + - 5 17 Acute colitis III + - 6 7 AcutecolitisI - + 14 Acute colitis III T T + 30 Normal 7 ? Acute colitis II T + 8 6 AcutecolitisI T T + + 30 Normal T + + + 9 ? Acute colitis II T + + 10 6 Acute colitis I T + 11 3 Acute colitis I T T + 12 10 Acute colitis II T T + 13 ? Acute colitis III T - + + 14 >7<14 Acute colitis II T T + + + 15 ? Acute colitis II T T 16 ? Acute colitis II T 17 14 Acute colitis II T T + 18 ? Normal T - + + 19 >7<14 Acute colitis II T + + + 20 ? Acute colitis II + 21 ? Acute colitis I T + 22 >14 Normal http://gut.bmj.com/ I = early stage, II = late stage, III = residual stage, T = increased, ? = unknown.

EARLY STAGES (FIVE PATIENTS) qand tend to be more pronounced in the distal parts In the first seven days after the onset of bloody of the colon (Fig. 3). diarrhoea histological changes are evident and diagnostic for acute infectious colitis. Epithelial LATE STAGE (12 PATIENTS) on September 30, 2021 by guest. Protected copyright. changes are conspicuous. The surface epithelium is From seven to 14 days after the beginning of infiltrated by neutrophils and superficial ulcerations symptoms the inflammation gradually subsides and are present. Often the surface is covered with the histological findings become less characteristic. exudate. Transmigration of granulocytes in the crypt The epithelium shows features of regeneration: epithelium gives rise to cryptitis (incipient crypt large pale nuclei with prominent nucleoli and there abscesses) that may ultimately result in crypt absces- is increased mitotic activity. Transmigration of ses: crypts filled with granulocytes lined by a flat neutrophils in crypts is either absent or may be seen degenerated epithelium. There usually is mucin focally adjacent to normal crypts giving the inflam- depletion of the epithelium. The lamina propria mation a discontinuous appearance. The lamina shows oedema and is diffusely infiltrated with propria is oedematous especially in the upper layers granulocytes, histiocytes, plasma cells, and lympho- and contains an inflammatory infiltrate consisting cytes. Granulocytes and histiocytes predominate. mainly or solely of mononuclear cells, especially in Occasionally the histiocytic component of the infil- the deeper layers (basal cryptitis). Lymphoid hyper- trate is very pronounced and gives the infiltrate a plasia may be found and crypt architecture is not granulomatous appearance. Granulomata with giant distorted. cells, however, were never observed. Apart from separation of crypts because of oedema and loss of RESIDUAL STAGE (THREE PATIENTS) crypts, crypt architecture is well preserved. The After 14 days inflammatory changes are minimal or pathological changes are confined to the mucosa absent. There may be a slight increase of lympho- Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from

948 van Spreeuwel, Duursma, Meijer, Bax, Rosekrans, and Lindeman

Campylobcter Control Crohn's Ulcerative Campylobacter Controls Crohn's Ulcerative colitis n = 22 n_10 disease of colitis colitis n=22 n=10 disease o colitis 200 the colon nz10 the colon n-10 n=10 (+ix 276) n=10 ILw,nnj . 1 x-= Patient with ulcerative colitis and superimposed ; x= Patient with ulcerative campylobacter colitis colitis and superimposed 150- campylobacter colitis 150- 3

0 100- 100- x~~~~~~~~~~~

5~~~~~~~~~~~~~~~~~~~~~~

it 50- x L 50-

so GAM 6

O0 A MG A MG A MG A M G O-1 Fig. 1 Numbers ofimmunoglobulin containing cells per Fig. 2 Numbers ofimmunoglobulin containing cells per

0-1 mm2 lamina propria ± standard deviation. 0 5 mm muscularis mucosae ± standard deviation. http://gut.bmj.com/ cytes and plasma cells in the lamina propria and tive colitis with respect to the number of IgG crypts may not reach to the muscularis mucosae. In containing cells. The number of IgG containing cells one patient we observed branching of crypts. is significantly lower in Campylobacter colitis compared with Crohn's colitis and ulcerative colitis, IMMUNOHISTOCHEMICAL FINDINGS both the number per 0-5 mm muscularis mucosae on September 30, 2021 by guest. Protected copyright. The numbers of IgA, IgM and IgG containing cells (p=001 and 0-00007 respectively) and the number are listed in Figs. 1 and 2. IgE containing cells were per 0-1 mm2 lamina propria (p=0002 and 0-00003 observed sporadically and therefore not included. respectively) differs significantly. Immunohistoche- The total number of immunoglobulin containing mical staining with Campylobacter antiserum gave cells is increased in Campylobacter colitis compared positive results in one or more biopsies in 19 out of with controls. This increase is because of IgA and 22 patients. The results with immunofluorescence IgM containing cells, both the number per 05 mm were equal to that obtained by immunoperoxidase. muscularis mucosae (p=00002 and p=003 respec- Positive staining was found in the glycocalyx (Fig. tively) and the number per 0-1 mm2 lamina propria 4), in the surface epithelium especially goblet cells (p=0.0005 and p=0001 respectively) differs signifi- and focally in the lamina propria. Occasionally cantly. The number of IgG containing cells per 0.1 positive staining was also found in crypt epithelium mm2 lamina propria was lower in Campylobacter and in histiocytes in the lamina propria. Positive colitis (p=0.01) compared with controls but this is immunohistochemical results did not correlate probably due to oedema of the lamina propria as the quantitatively or qualitatively with the degree of number of IgG containing cells per 0-5 mm muscu- inflammation and were also obtained in one patient laris mucosae does not differ. Biopsies of patients that showed no histological abnormalities. with Campylobacter colitis differ significantly from In order to confirm the immunohistochemical biopsies of patients with Crohn's colitis and ulcera- findings with Campylobacter antiserum serial sec- Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from

Campylobacter colitis: histological immunohistochemilcal and ultrastructural findings 949 .ni*'8 ¾.tt'i * N tions of the biopsies of four patients giving positive results were examined ultrastructurally. These sections were compared with the ultrastructural picture of a Campylobacter jejuni culture (Fig. 5). In all these biopsies characteristic features of Campylo- bacter could be demonstrated in either the glycocalyx (Fig. 6) overlying the surface epithelium or in the cytoplasma of the surface epithelium (Fig. 7) including Goblet cells. Discussion In this study we have shown that Campylobacter infection produces the histological picture of acute infectious colitis and that the number of immunoglobulin containing cells in the colonic biopsies of these patients differs from those of patients with chronic inflammatory bowel disease with regard to the number of IgG containing cells. In addition we have been able to show Campylobacter immunohistochemically in colonic biopsies in 19 out of 22 patients with positive stool cultures for Campylobacter jejuni. Campylobacter colitis should be distinguished from other bacillary dysenteries and from active Fig. 3 Colonic biopsy (seven days after the onset of chronic inflammatory bowel disease because it may symptoms) showing diffuse infiltration ofthe lamina mimic these diseases both clinically and endoscopic- propria with granulocytes, histiocytes, lymphocytes and ally. Stool cultures and colonoscopy with biopsy are erosion and important tools in the differential diagnosis. Histo- plasma cells, focal cryptitis. http://gut.bmj.com/ on September 30, 2021 by guest. Protected copyright.

Fig. 4 Indirect immunofluorescence with Campylobacter antiserum showingpositive staining in the glycocalyx overlying the surface epithelium x 400. Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from

950 van Spreeuwel, Duursma, Meijer, Bax, Rosekrans, and Lindeman http://gut.bmj.com/ Fig. 5-Electron micrograph ofa Campylobacter culture g. trcuralpicture ofthe glycocalyx showing a fixed in formalin and embedded in epon x 10 000. Showing transversal section of Campylobacter microorganisms x Campylobacter microorganisms (arrowhead). 10 000 (arrowhead).

logically considerable overlap with chronic inflam- bacter colitis retrospectively. Although most matory bowel disease exists and differentiation may patients recover from their illness within one week be difficult especially in the late stages. the median duration of excretion of microorganisms Determination of the number of IgA, IgM, and in untreated cases is two to three weeks,18 this may on September 30, 2021 by guest. Protected copyright. IgG containing cells may be helpful in the differen- explain by Campylobacter can still be shown tial diagnosis between Campylobacter colitis and immunohistochemically after the patient has re- active chronic inflammatory bowel disease because covered. Because immunohistochemistry with the number of IgG containing cells is normal in the Campylobacter antiserum yields positive results in former whereas the number of IgG containing cells 19 out of 22 patients with stool cultures positive for is increased in active chronic inflammatory bowel Campylobacterjejuni it can be considered a sensitive disease. 16 As these findings are non-specific the technique. histological diagnosis of Campylobacter colitis ulti- The mechanism by which Campylobacter causes mately relies on demonstration of Campylobacter disease is not known.'9 Although the organism was microorganisms immunohistochemically. Campylo- originally isolated from the blood,20 positive blood bacter colitis cannot be distinguished histologically cultures have only been reported occasionally.2' from other bacillary dysenteries nor is it possible to Campylobacter jejuni has been shown to invade diagnose Campylobacter infection histologically chicken embryo cells in vitro21 but evidence for when it is superimposed on chronic inflammatory tissue invasion in man is lacking. Alternatively tissue bowel disease.17 Immunohistochemistry with Cam- damage could be produced by toxins. Our findings pylobacter antiserum may prove useful in these with Campylobacter antiserum that were confirmed cases. It also makes it possible to diagnose Campylo- ultrastructurally clearly show that Campylobacter Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from

Campylobacter colitis: histological immunohistochemical and ultrastructural findings 951

Reller LB, Wang WLL. Campylobacter enteritis: clinical and epidemiological features. Ann Intern Med 1979; 91: 179-85. 2 Drake AA, Gilchrist MJR, Washington JA, Huizenga KA, Van Scoy RE. Diarrhea due to Campylobacter fetus subspecies jejuni. A clinical review of 63 cases. Mayo Clin Proc 1981; 56: 414-23. 3 Skirrow MB. Campylobacter enteritis: a "new" disease. Br Med J 1979; 2: 9-11. 4 McKinley MJ, Taylor M, Sangree MH. Toxic megacolon with Campylobacter colitis. Conn Med 1980; 44: I 496-7. V~~~~~~~~~~~~ 5 Mertens A, De Smet M. Campylobacter cholecystitis. Lancet 1979; I: 1091-2. 6 Lambert ME, Schofield PF, Ironside AG, Mandal BK. Campylobacter colitis. Br Med J 1979; 1: 857-9. 7 Willoughby CP, Piris J, Truelove SC. Campylobacter colitis. J Clin Pathol 1979; 32: 986-9. 8 Price AB, Jewkes J, Sanderson PJ. Acute diarrhoea: Campylobacter colitis and the role of rectal biopsy. J Clin Pathol 1979; 32: 990-7. 9 Dickinson RJ, Gilmour MM, McClelland DBH. Rectal biopsy in patients presenting to an infectious disease unit with diarrhoeal disease. Gut 1979; 20: 141-8. 10 Vesterby A, Baandrup U, Jacobsen NO, Albertsen K. Campylobacter enterocolitis, an important differential diagnosis in ulcerative colitis. Acta Pathol Microbiol Immunol Scand 1983; Sect. A 91: 31-33. 11 Kumar NB, Nostrant TT, Appelman HD. The histo- pathological spectrum of acute self-limited colitis (acute infectious-type colitis). Am J Surg Pathol 1982; 6: 523-9. 12 Bosman FT, Lindeman J, Kuipers G, van der Wal AM, http://gut.bmj.com/ Kreuning J. The influence of fixation on immuno- Fig. 7 Electron micrograph ofmoderatelypreserved reactivity of plasma cells in routinely processed intes- Goblet@. *;cell ...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... (the cell wall:t.i.j!, is marked by small arrowheads) tinal biopsy specimens. Histochemistry 1977; 53: 57-62. showing several transversal sections ofCampylobacter 13 Sternberger L. Immunohistochemistry. Englewood microorganisms (large arrowheads) x 6000. Cliffs, NJ: Prentice Hall 1979; 53. 14 Brandtzaeg P, Baklien K, Fausa 0, Hoel PS. Immuno- histochemical characterisation of local immunoglobulin invades the colonic mucosa. Recently it has been formation in ulcerative colitis. Gastroenterology 1974; on September 30, 2021 by guest. Protected copyright. shown that Campylobacterjej'uni also may produce a 66: 1123-36. 22 15 Lauwers S, Vlaes L, Butzler JP. Campylobacter cholera like enterotoxin. serotyping and epidemiology. Lancet 1981; I: 158-9. It is concluded that Campylobacter infections 16 Rosekrans PCM, Meijer CJLM, Van der Wal AM, produce the histological picture of acute infectious Cornelisse CJ, Lindeman J. Immunoglobulin contain- colitis. The histological diagnosis of acute infectious ing cells in inflammatory bowel disease of the colon: a colitis, however, is difficult especially in the later morphometric and immunohistochemical study. Gut stages. Immunohistochemical demonstration of im- 1980; 21: 941-7. munoglobulin containing cells and Campylobacter 17 Day DW, Mandal BK, Morson BC. The rectal biopsy microorganisms contributes significantly to the his- appearances in salmonella colitis. Histopathology 1978; colitis. 2: 117-31. tological diagnosis of Campylobacter 18 Blaser MJ, Reller LB. Campylobacter enteritis. N Engl J Med 1981; 305: 1444-52. The authors wish to express their gratitude to Mrs P 19 [Editorial]. Lancet 1982; 2: 1437-8. P Blommers and Mrs R JJ R Scholte who prepared 20 King EO. Human infections with vibrio fetus and a the manuscript. closely related vibrio. J Infect Dis 1957; 101: 119-28. 21 Butzler JP, Skirrow MB. Campylobacter enteritis. Clin Gastroenterol 1979; 8: 737-65. References 22 Ruiz-Palacios GM, Torres J, Torres N, Escamilla E, Ruiz-Palacios B. Cholera-like enterotoxin produced by 1 Blaser MJ, Berkowitz ID, La Force FM, Cravens J, Campylobacter jejuni. Lancet 1983; 2: 250-2.